Human food and animal feed derived from many grains are deficient in a number of the ten essential amino acids which are required in the animal diet. Of importance in improving food or feed value of the cereal grain crops is the ability to manipulate genes encoding proteins that contain high levels of essential amino acids. For example, to be nutritionally adequate and support optimal growth of chickens, corn-soybean meal poultry feed is generally supplemented with synthetic methionine or a methionine analog. The development of lines of corn which supply higher levels of methionine can reduce the need for methionine supplements.
Lysine, an amino acid essential in the diets of humans and monogastric animals, is among the three most limiting amino acids in most of the staple crops, the cereals in particular. Consequently, grain-based diets must be supplemented with synthetic lysine or with lysine-containing oil-seed protein meals. Further, since most oilseed meals are themselves inadequate lysine sources, balancing the feed mixture for lysine frequently results in meals which are too high in other, less desirable nutrients. Therefore, a method to increase the lysine content of either the cereal grains or the oilseed crops of both would result in significant added nutritional value, as well as a significant cost savings to end users such as the swine and poultry producers.
Plant breeders have long been interested in using naturally occurring variations to improve protein quality and quantity in crop plants. Maize lines containing higher than normal levels of lysine (70%) have been identified (Mertz et al., Science, 145, 279 (1964); Mertz et al., Science, 150, 1469 (1965)). However, these lines which incorporate a mutant gene, opaque-2, exhibit poor agronomic qualities, such as reduction in yield, slower drying and increased storage problems, and thus are not commercially useful (Deutscher, Adv. Exp. Medicine and Biology, 105, 281 (1978)). Quality Protein Maize (QPM) bred at CIMMYT using the opaque-2 and sugary-2 genes and associated modifiers has a hard endosperm and enriched levels of lysine and tryptophan in the kernels (S. K. Vasal et al., Proceedings of the 3rd seed protein symposium, Gatersleben, Aug. 31–Sep. 2, 1983). However, the gene pools represented in the QPM lines are tropical and subtropical. Quality Protein Maize is a genetically complex trait and the existing lines are not easily adapted to the dent germplasm in use in the United States, thus preventing the adoption of QPM by corn breeders.
The amino acid content of seeds is determined primarily (90–99%) by the amino acid composition of the proteins in the seed and to a lesser extent (1–10%) by the free amino acid pools. The quantity of total protein in seeds varies from about 10% of the dry weight in cereals to 20–40% of the dry weight of legumes. Much of the protein-bound amino acids is contained in the seed storage proteins which are synthesized during seed development and which serve as a major nutrient reserve following germination. In many seeds the storage proteins account for 50% or more of the total protein.
To improve the amino acid composition of seeds, genetic engineering technology is being used to isolate, and express genes for storage proteins in transgenic plants. For example, a gene from Brazil nut for a seed 2S albumin composed of 26% sulfur-containing amino acids has been isolated and expressed in the seeds of transformed tobacco under the control of the regulatory sequences from a bean phaseolin storage protein gene. The accumulation of the sulfur-rich protein in the tobacco seeds resulted in an up to 30% increase in the level of methionine in the seeds (Altenbach et al., Plant Mol. Biol., 13, 513 (1989)). However, the potential for allergic reactions in humans exposed to this heterologous protein has limited use of this approach.
The E. coli dapA gene encodes a DHDPS enzyme that is about 20-fold less sensitive to inhibition by lysine than a typical plant DHDPS enzyme, e.g., wheat germ DHDPS. The E. coli dapA gene has been linked to the 35S promoter of Cauliflower Mosaic Virus and a plant chloroplast transit sequence. The chimeric gene was introduced into tobacco cells via transformation and shown to cause a substantial increase in free lysine levels in leaves (Glassman et al., U.S. Pat. No. 5,258,300; Shaul et al., Plant Jour., 2, 203 (1992); Galili et al., EPO Patent Appl. 91119328.2 (1992)).
The 10 kD-zein storage protein is produced in the endosperm of the maize kernel and contains extremely high levels of methionine (22.5%). It is encoded by the Zps10/(22) gene (M. S. Benner et al., Theor. Appl. Genet., 78 761 (1989). Thus, increased expression of this gene can be used to increase the methionine content of corn. Introduction of exogenous genes into monocots such as the cereal plants has proven to be scientifically more challenging than the transformation of dicots. Lundquist et al. (U.S. Pat. No. 5,508,468) disclose production of transgenic maize expressing the maize 10 kD-zein gene by microprojectile bombardment of regenerable maize cells with a chimeric construct comprising the coding region of this gene. However, from a practical standpoint, approaches based on increasing the total protein content of maize are limited by the nitrogen requirements, cost and lower productivity, and the actual pathogen resistance and hardiness of the resulting high protein variants.
Therefore, a need exists for methods to selectively increase the content of amino acids such as by increasing high methionine or high lysine proteins in maize and other cereal (monocot) plants.